Over the past few decades the art of protein sequence analysis has been propelled by advances in the field of mass spectrometry (Domon et al., Review—Mass spectrometry and protein analysis. Science, 2006. 312(5771): 212-217; Ashcroft, A. E., Protein and peptide identification: the role of mass spectrometry in proteomics. Natural Product Reports, 2003. 20(2):202-215; Mann et al., Analysis of proteins and proteomes by mass spectrometry. Annual Review of Biochemistry, 2001. 70:437-473; and Coon et al., Tandem mass spectrometry for peptide and protein sequence analysis. Biotechniques, 2005. 38(4): 519, 521, 523). At the core of these technologies is tandem mass spectrometry (MS/MS)—the process of peptide or protein ion dissociation with subsequent m/z analysis. As such, effective peptide ion fragmentation techniques are essential. The likelihood of successfully identifying a selected peptide or protein is primarily dependent upon the extent and quality of backbone fragmentation produced.
For years collision-activated dissociation (CAD) has been the primary method of implementing MS/MS. During CAD, a population of selected peptide cations undergoes collisions with an inert bath gas. The generated internal energy is distributed across the backbone of the peptide to induce cleavage of the weakest bonds (Zubarev et al., Electron capture dissociation of multiply charged protein cations. A nonergodic process. Journal of the American Chemical Society, 1998. 120(13):3265-3266). For peptide cations the protonated amide bonds are weakened and, in general, are favored for cleavage upon CAD. The CAD process, however, tends to fail in this regard when the target peptide contains: (1) a post-translational modification (PTM) that fragments through a lower energy pathway (e.g., phosphorylation, glycosylation, sulfonation, etc.), (2) certain amino acids, especially those that inhibit random protonation of the peptide backbone, and (3) more than ˜15 amino acids (Dongre et al., Influence of peptide composition, gas-phase basicity, and chemical modification on fragmentation efficiency: Evidence for the mobile proton model. Journal of the American Chemical Society, 1996. 118(35):8365-8374; and Wysocki et al., Mass spectrometry of peptides and proteins. Methods, 2005. 35(3):211-222).
The shortcomings in tandem MS (i.e., CAD) can be eliminated by fragmentation technology using electron based dissociation methods, such as electron capture dissociation (ECD) or electron transfer dissociation (ETD). ECD and ETD are non-ergodic techniques that rely on either the capture or transfer of an electron to the peptide cation precursor to impart fragmentation (Zubarev et al., Towards an understanding of the mechanism of electron-capture dissociation: a historical perspective and modern ideas. European Journal of Mass Spectrometry, 2002. 8(5):337-349; Ge et al., Top down characterization of larger proteins (45 kDa) by electron capture dissociation mass spectrometry. Journal of the American Chemical Society, 2002. 124(4):672-678; Cooper et al., The role of electron capture dissociation in biomolecular analysis. Mass Spectrometry Reviews, 2005. 24(2):201-222; Syka et al., Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry. Proceedings of the National Academy of Sciences of the United States of America, 2004. 101 (26):9528-9533; Coon et al., Electron transfer dissociation of peptide anions. Journal of the American Society for Mass Spectrometry, 2005. 16(6):880-882; Coon, J. J., et al., Protein identification using sequential ion/ion reactions and tandem mass spectrometry. Proceedings of the National Academy of Sciences of the United States of America, 2005. 102(27):9463-9468; and Mikesh et al., The utility of ETD mass spectrometry in proteomic analysis. Biochimica Et Biophysica Acta-Proteins and Proteomics, 2006. 1764(12):1811-1822).
Rather than using collisions, ETD reacts the selected peptide cations with anions of fluoranthene (or other negatively charged small molecules). This reaction proceeds by transfer of an electron from the fluoranthene anion to the peptide (an ion/ion reaction). The added electron causes the peptide to break randomly between each amino acid. Once the peptide is fragmented, the masses of each fragment are then recorded and used with the mass of the parent peptide to analyze the peptide. Unlike CAD, ETD causes cleavage of a different backbone bond to produce c and z-type fragment ions, rather than the b and y-type fragments generated by CAD. ETD can be considered a derivative of electron capture dissociation ECD which uses free electrons rather than anions to induce the same fragmentation pathways.
Whether performed in an ion cyclotron resonance cell of a Fourier transform mass spectrometer (FT-ICR-MS, ECD) or in a RF quadrupole ion trap (QIT, ETD), these electron based dissociation methods induce random backbone cleavage with little regard for the presence of PTMs, amino acid composition, or the number of amino acids in the sequence. Of course, the electron based methods are not without their own limitations. Early work employing ECD and recent large-scale experiments with ETD indicate that precursor cation charge density may be the most critical parameter in determining a successful sequencing outcome (Good et al., Performance characteristics of electron transfer dissociation mass spectrometry. Molecular & Cellular Proteomics, 2007. 6(11):1942-1951). For ETD, percent fragmentation—defined as the number of observed c and z-type fragments divided by the number possible—decreases with increasing precursor residue per charge ratio. Since the amino acids have similar residue mass values and peptides are collections of amino acids, precursor mass-to-charge (m/z) can approximate the residue per charge ratio. Precursor peptide cations with m/z values above ˜900—regardless of z—have a low probability of generating sufficient direct backbone fragmentation for sequence assignment.
Previous experiments have described attaching quaternary amines to peptides in order to increase the charge state for mass spectroscopy (published PCT application WO 2007/109,292, published on Sep. 27, 2007). While the quaternary amines successfully increased the charge state of the peptide, the resulting mass spectrometry spectra were too complex and chaotic to yield useful information. Additionally, tagging peptides with quaternary amines reduced the ability to purify the peptides by chromatography.
Previous experiments have also described techniques to coax the conversion of the non-dissociated EC/ET products to c and z-type fragments from precursor peptide cations (Swaney et al., Supplemental activation method for high-efficiency electron-transfer dissociation of doubly protonated peptide precursors. Analytical Chemistry, 2007. 79(2):477-485). One drawback of that approach is that the resultant products often undergo hydrogen atom rearrangement to render the c and z-type products either one Da lighter or heavier, respectively. These peaks are superposed onto the isotopic distributions of the directly produced ETD fragments and can be problematic during sequence assignment.